COVID-19: A complex disease with a unique metabolic signature.

Plasma of COVID-19 patients contains a strong metabolomic/lipoproteomic signature, revealed by the NMR analysis of a cohort of >500 patients sampled during various waves of COVID-19 infection, corresponding to the spread of different variants, and having different vaccination status. This composite signature highlights common traits of the SARS-CoV-2 infection. The most dysregulated molecules display concentration trends that scale with disease severity and might serve as prognostic markers for fatal events. Metabolomics evidence is then used as input data for a sex-specific multi-organ metabolic model. This reconstruction provides a comprehensive view of the impact of COVID-19 on the entire human metabolism. The human (male and female) metabolic network is strongly impacted by the disease to an extent dictated by its severity. A marked metabolic reprogramming at the level of many organs indicates an increase in the generic energetic demand of the organism following infection. Sex-specific modulation of immune response is also suggested.

Profiling metabolites and lipoproteins in COMETA, an Italian cohort of COVID-19 patients.

Metabolomics and lipidomics have been used in several studies to define the biochemical alterations induced by COVID-19 in comparison with healthy controls. Those studies highlighted the presence of a strong signature, attributable to both metabolites and lipoproteins/lipids. Here, 1H NMR spectra were acquired on EDTA-plasma from three groups of subjects: i) hospitalized COVID-19 positive patients (≤21 days from the first positive nasopharyngeal swab); ii) hospitalized COVID-19 positive patients (>21 days from the first positive nasopharyngeal swab); iii) subjects after 2-6 months from SARS-CoV-2 eradication. A Random Forest model built using the EDTA-plasma spectra of COVID-19 patients ≤21 days and Post COVID-19 subjects, provided a high discrimination accuracy (93.6%), indicating both the presence of a strong fingerprint of the acute infection and the substantial metabolic healing of Post COVID-19 subjects. The differences originate from significant alterations in the concentrations of 16 metabolites and 74 lipoprotein components. The model was then used to predict the spectra of COVID-19>21 days subjects. In this group, the metabolite levels are closer to those of the Post COVID-19 subjects than to those of the COVID-19≤21 days; the opposite occurs for the lipoproteins. Within the acute phase patients, characteristic trends in metabolite levels are observed as a function of the disease severity. The metabolites found altered in COVID-19≤21 days patients with respect to Post COVID-19 individuals overlap with acute infection biomarkers identified previously in comparison with healthy subjects. Along the trajectory towards healing, the metabolome reverts back to the "healthy" state faster than the lipoproteome.

Machine Learning-Enhanced Quantum Chemistry-Assisted Refinement of the Active Site Structure of Metalloproteins.

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.

Exploring the Effects of Probiotic Treatment on Urinary and Serum Metabolic Profiles in Healthy Individuals.

Probiotics are live microorganisms that confer health benefits when administered in adequate amounts. They are used to promote gut health and alleviate various disorders. Recently, there has been an increasing interest in the potential effects of probiotics on human physiology. In the presented study, the effects of probiotic treatment on the metabolic profiles of human urine and serum using a nuclear magnetic resonance (NMR)-based metabonomic approach were investigated. Twenty-one healthy volunteers were enrolled in the study, and they received two different dosages of probiotics for 8 weeks. During the study, urine and serum samples were collected from volunteers before and during probiotic supplementation. The results showed that probiotics had a significant impact on the urinary and serum metabolic profiles without altering their phenotypes. This study demonstrated the effects of probiotics in terms of variations of metabolite levels resulting also from the different probiotic posology. Overall, the results suggest that probiotic administration may affect both urine and serum metabolomes, although more research is needed to understand the mechanisms and clinical implications of these effects. NMR-based metabonomic analysis of biofluids is a powerful tool for monitoring host-gut microflora dynamic interaction as well as for assessing the individual response to probiotic treatment.

Nuclear Magnetic Resonance-Based Metabolomics to Predict Early and Late Adverse Outcomes in Ischemic Stroke Treated with Intravenous Thrombolysis.

Metabolic perturbations and inflammatory mediators play a fundamental role in both early and late adverse post-acute ischemic stroke outcomes. Using data from the observational MAGIC (MArker bioloGici nell'Ictus Cerebrale) study, we evaluated the effect of 130 serum metabolic features, using a nuclear magnetic spectroscopy approach, on the following outcomes: hemorrhagic transformation at 24 h after stroke, non-response to intravenous thrombolytic treatment with the recombinant tissue plasminogen activator (rt-PA), and the 3 month functional outcome. Blood circulating metabolites, lipoproteins, and inflammatory markers were assessed at the baseline and 24 h after rt-PA treatment. Adjusting for the major determinants for unfavorable outcomes (i.e., age, sex, time onset-to-treatment, etc.), we found that acetone and 3-hydroxybutyrate were associated with symptomatic hemorrhagic transformation and with non-response to rt-PA; while 24 h after rt-PA, levels of triglycerides high-density lipoprotein (HDL) and triglycerides low-density lipoprotein (LDL) were associated with 3 month mortality. Cholesterol and phospholipids levels, mainly related to smaller and denser very low-density lipoprotein (VLDL) and LDL subfractions were associated with 3 month poor functional outcomes. We also reported associations between baseline 24 h relative variation (Δ) in VLDL subfractions and ΔC-reactive protein, Δinterleukin-10 levels with hemorrhagic transformation. All observed metabolic changes reflect a general condition of energy failure, oxidative stress, and systemic inflammation that characterize the development of adverse outcomes.

Molecular Engineering of Self-Immolative Bioresponsive MR Probes.

Real-time detection of bio-event in whole animals provides essential information for understanding biological and therapeutic processes. Magnetic resonance (MR) imaging represents a non-invasive approach to generating three-dimensional anatomic images with high spatial-temporal resolution and unlimited depth penetration. We have developed several self-immolative enzyme-activatable agents that provide excellent in vivo contrast and function as gene expression reporters. Here, we describe a vast improvement in image contrast over our previous generations of these bioresponsive agents based on a new pyridyl-carbamate Gd(III) complex. The pyridyl-carbamate-based agent has a very low MR relaxivity in the "off-state" (r1 = 1.8 mM-1 s-1 at 1.41 T). However, upon enzymatic processing, it generates a significantly higher relaxivity with a Δr1 = 106% versus Δr1 ∼ 20% reported previously. Single X-ray crystal and nuclear magnetic relaxation dispersion analyses offer mechanistic insights regarding MR signal enhancement at the molecular scale. This work demonstrates a pyridyl-carbamate-based self-immolative molecular platform for the construction of enzymatic bio-responsive MR agents, which can be adapted to a wide range of other targets for exploring stimuli-responsive materials and biomedical applications.

Novel Nanogels Loaded with Mn(II) Chelates as Effective and Biologically Stable MRI Probes.

Here it is described nanogels (NG) based on a chitosan matrix, which are covalently stabilized by a bisamide derivative of Mn-t-CDTA (t-CDTA = trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid). the Mn(II) complex acts both as a contrast medium and as a cross-linking agent. These nanogels are proposed as an alternative to the less stable paramagnetic nanogels obtained by electrostatic interactions between the polymeric matrix and paramagnetic Gd(III) chelates. The present novel nanogels show: i) relaxivity values seven times higher than that of typical monohydrated Mn(II) chelates at the clinical fields, thanks to the combination of a restricted mobility of the complex with a fast exchange of the metal-bound water molecule; ii) high stability of the formulation over time at pH 5 and under physiological conditions, thus excluding metal leaking or particles aggregation; iii) good extravasation and accumulation, with a maximum contrast achieved at 24 h post-injection in mice bearing subcutaneous breast cancer tumor; iv) high T1 contrast (1 T) in the tumor 24 h post-injection. These improved properties pave the way for the use of these paramagnetic nanogels as promising magnetic resonance imaging (MRI) probes for in vitro and in vivo preclinical applications.

Metabolomic/lipidomic profiling of COVID-19 and individual response to tocilizumab.

The current pandemic emergence of novel coronavirus disease (COVID-19) poses a relevant threat to global health. SARS-CoV-2 infection is characterized by a wide range of clinical manifestations, ranging from absence of symptoms to severe forms that need intensive care treatment. Here, plasma-EDTA samples of 30 patients compared with age- and sex-matched controls were analyzed via untargeted nuclear magnetic resonance (NMR)-based metabolomics and lipidomics. With the same approach, the effect of tocilizumab administration was evaluated in a subset of patients. Despite the heterogeneity of the clinical symptoms, COVID-19 patients are characterized by common plasma metabolomic and lipidomic signatures (91.7% and 87.5% accuracy, respectively, when compared to controls). Tocilizumab treatment resulted in at least partial reversion of the metabolic alterations due to SARS-CoV-2 infection. In conclusion, NMR-based metabolomic and lipidomic profiling provides novel insights into the pathophysiological mechanism of human response to SARS-CoV-2 infection and to monitor treatment outcomes.

Interhelical interactions within the STIM1 CC1 domain modulate CRAC channel activation.

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

NMR-Based Metabolomics to Evaluate Individual Response to Treatments.

The aim of this chapter is to highlight the various aspects of metabolomics in relation to health and diseases, starting from the definition of metabolic space and of how individuals tend to maintain their own position in this space. Physio-pathological stimuli may cause individuals to lose their position and then regain it, or move irreversibly to other positions. By way of examples, mostly selected from our own work using 1H NMR on biological fluids, we describe the effects on the individual metabolomic fingerprint of mild external interventions, such as diet or probiotic administration. Then we move to pathologies (such as celiac disease, various types of cancer, viral infections, and other diseases), each characterized by a well-defined metabolomic fingerprint. We describe the effects of drugs on the disease fingerprint and on its reversal to a healthy metabolomic status. Drug toxicity can be also monitored by metabolomics. We also show how the individual metabolomic fingerprint at the onset of a disease may discriminate responders from non-responders to a given drug, or how it may be prognostic of e.g., cancer recurrence after many years. In parallel with fingerprinting, profiling (i.e., the identification and quantification of many metabolites and, in the case of selected biofluids, of the lipoprotein components that contribute to the 1H NMR spectral features) can provide hints on the metabolic pathways that are altered by a disease and assess their restoration after treatment.

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates.

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.

Serum or Plasma (and Which Plasma), That Is the Question.

Blood derivatives are the biofluids of choice for metabolomic clinical studies since blood can be collected with low invasiveness and is rich in biological information. However, the choice of the blood collection tubes has an undeniable impact on the plasma and serum metabolic content. Here, we compared the metabolomic and lipoprotein profiles of blood samples collected at the same time and place from six healthy volunteers but using different collection tubes (each enrolled volunteer provided multiple blood samples at a distance of a few weeks/months): citrate plasma, EDTA plasma, and serum tubes. All samples were analyzed via nuclear magnetic resonance spectroscopy. Several metabolites showed statistically significant alterations among the three blood matrices, and also metabolites' correlations were shown to be affected. The effects of blood collection tubes on the lipoproteins' profiles are relevant too, but less marked. Overcoming the issue associated with different blood collection tubes is pivotal to scale metabolomics and lipoprotein analysis at the level of epidemiological studies based on samples from multicenter cohorts. We propose a statistical solution, based on regression, that is shown to be efficient in reducing the alterations induced by the different collection tubes for both the metabolomic and lipoprotein profiles.

Association of Plasma Metabolites and Lipoproteins with Rh and ABO Blood Systems in Healthy Subjects.

This study investigated the associations between the levels of 27 plasma metabolites, 114 lipoprotein parameters, determined using nuclear magnetic resonance spectroscopy, and the ABO blood groups and the Rhesus (Rh) blood system in a cohort of n = 840 Italian healthy blood donors of both sexes. We observed good multivariate discrimination between the metabolomic and lipoproteomic profiles of subjects with positive and negative Rh. In contrast, we did not observe significant discrimination for the ABO blood group pairwise comparisons, suggesting only slight metabolic differences between these group-specific metabolic profiles. We report univariate associations (P-value < 0.05) between the subfraction HDL1 related to Apo A1, the subfraction HDL2 related to cholesterol and phospholipids, and the particle number of LDL2 related to free cholesterol, cholesterol, phospholipids, and Apo B and the ABO blood groups; we observed association of the lipid main fraction LDL4 related to free cholesterol, triglycerides, and Apo B; creatine; the particle number of LDL5; the subfraction LDL5 related to Apo B; the particle number of LDL4; and the subfraction LDL4 related to Apo B with Rh blood factors. These results suggest blood group-dependent (re)shaping of lipoprotein metabolism in healthy subjects, which may provide relevant information to explain the differential susceptibility to certain diseases observed in different blood groups.

Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm.

Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.

Not only manganese, but fruit component effects dictate the efficiency of fruit juice as an oral magnetic resonance imaging contrast agent.

Several fruit juices are used as oral contrast agents to improve the quality of images in magnetic resonance cholangiopancreatography. They are often preferred to conventional synthetic contrast agents because of their very low cost, natural origin, intrinsic safety, and comparable image qualities. Pineapple and blueberry juices are the most employed in clinical practice due to their higher content of manganese(II) ions. The interest of pharmaceutical companies in these products is testified by the appearance in the market of fruit juice derivatives with improved contrast efficacy. Here, we investigate the origin of the contrast of blueberry juice, analyze the parameters that can effect it, and elucidate the differences with pineapple juice and manganese(II) solutions. It appears that, although manganese(II) is the paramagnetic ion responsible for the contrast, it is the interaction of manganese(II) with other juice components that modulates the efficiency of the juice as a magnetic resonance contrast agent. On these grounds, we conclude that blueberry juice concentrated to the same manganese concentration of pineapple juice would prove a more efficient contrast agent than pineapple juice.

High Relaxivity with No Coordinated Waters: A Seemingly Paradoxical Behavior of [Gd(DOTP)]5- Embedded in Nanogels.

Nanogels (NGs) obtained by electrostatic interactions between chitosan and hyaluronic acid and comprising paramagnetic Gd chelates are gaining increasing attention for their potential application in magnetic resonance bioimaging. Herein, the macrocyclic complexes [Gd(DOTP)]5-, lacking metal-bound water molecules (q = 0), were confined or used as a cross-linker in this type of NG. Unlike the typical behavior of Gd complexes with q = 0, a remarkable relaxivity value of 78.0 mM-1 s-1 was measured at 20 MHz and 298 K, nearly 20 times greater than that found for the free complex. A careful analysis of the relaxation data emphasizes the fundamental role of second sphere water molecules with strong and long-lived hydrogen bonding interactions with the complex. Finally, PEGylated derivatives of nanoparticles were used for the first in vivo magnetic resonance imaging study of this type of NG, revealing a fast renal excretion of paramagnetic complexes after their release from the NGs.

The evolution of paramagnetic NMR as a tool in structural biology.

Paramagnetic NMR data contain extremely accurate long-range information on metalloprotein structures and, when used in the frame of integrative structural biology approaches, they allow for the retrieval of structural details to a resolution that is not achievable using other techniques. Paramagnetic data thus represent an extremely powerful tool to refine protein models in solution, especially when coupled to X-ray or cryoelectron microscopy data, to monitor the formation of complexes and determine the relative arrangements of their components, and to highlight the presence of conformational heterogeneity. More recently, theoretical and computational advancements in quantum chemical calculations of paramagnetic NMR observables are progressively opening new routes in structural biology, because they allow for the determination of the structure within the coordination sphere of the metal center, thus acting as a loupe on sites that are difficult to observe but very important for protein function.

Theoretical analysis of the long-distance limit of NMR chemical shieldings.

After some years of controversy, it was recently demonstrated how to obtain the correct long-distance limit [point-dipole approximation (PDA)] of pseudo-contact nuclear magnetic resonance chemical shifts from rigorous first-principles quantum mechanics [Lang et al., J. Phys. Chem. Lett. 11, 8735 (2020)]. This result confirmed the classical Kurland-McGarvey theory. In the present contribution, we elaborate on these results. In particular, we provide a detailed derivation of the PDA both from the Van den Heuvel-Soncini equation for the chemical shielding tensor and from a spin Hamiltonian approximation. Furthermore, we discuss in detail the PDA within the approximate density functional theory and Hartree-Fock theories. In our previous work, we assumed a relatively crude effective nuclear charge approximation for the spin-orbit coupling operator. Here, we overcome this assumption by demonstrating that the derivation is also possible within the fully relativistic Dirac equation and even without the assumption of a specific form for the Hamiltonian. Crucial ingredients for the general derivation are a Hamiltonian that respects gauge invariance, the multipolar gauge, and functional derivatives of the Hamiltonian, where it is possible to identify the first functional derivative with the electron number current density operator. The present work forms an important foundation for future extensions of the Kurland-McGarvey theory beyond the PDA, including induced magnetic quadrupole and higher moments to describe the magnetic hyperfine field.

Solid-state NMR methods for the characterization of bioconjugations and protein-material interactions.

Protein solid-state NMR has evolved dramatically over the last two decades, with the development of new hardware and sample preparation methodologies. This technique is now ripe for complex applications, among which one can count bioconjugation, protein chemistry and functional biomaterials. In this review, we provide our account on this aspect of protein solid-state NMR.

Differential Network Analysis Reveals Molecular Determinants Associated with Blood Pressure and Heart Rate in Healthy Subjects.

There is mounting evidence that subclinical nonpathological high blood pressure and heart rate during youth and adulthood steadily increase the risk of developing a cardiovascular disease at a later stage. For this reason, it is important to understand the mechanisms underlying the subclinical elevation of blood pressure and heart rate in healthy, relatively young individuals. In the present study, we present a network-based metabolomic study of blood plasma metabolites and lipids measured using nuclear magnetic resonance spectroscopy on 841 adult healthy blood donor volunteers, which were stratified for subclinical low and high blood pressure (systolic and diastolic) and heart rate. Our results indicate a rewiring of metabolic pathways active in high and low groups, indicating that the subjects with subclinical high blood pressure and heart rate could present latent cardiometabolic dysregulations.